Breast cancer is the leading cause of cancer-related deaths in women. Altered cellular functions of cancer cells lead to uncontrolled cellular growth and morphological changes. Cellular biomembranes are intimately involved in the regulation of cell signaling; however, they remain largely understudied. Phospholipids (PLs) are the main constituents of biological membranes and play important functional, structural and metabolic roles. The aim of this study was to establish if patterns in the PL profiles of mammary epithelial cells and breast cancer cells differ in relation to degree of differentiation and metastatic potential. For this purpose, PLs were analyzed using a lipidomic approach. In brief, PLs were extracted using Bligh and Dyer method, followed by a separation of PL classes by thin layer chromatography, and subsequent analysis by mass spectrometry (MS). Differences and similarities were found in the relative levels of PL content between mammary epithelial and breast cancer cells and between breast cancer cells with different levels of aggressiveness. When compared to the total PL content, phosphatidylcholine levels were reduced and lysophosphatydilcholines increased in the more aggressive cancer cells; while phosphatidylserine levels remained unchanged. MS analysis showed alterations in the classes of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, and phosphatidylinositides. In particular, the phosphatidylinositides, which are signaling molecules that affect proliferation, survival, and migration, showed dramatic alterations in their profile, where an increase of phosphatdylinositides saturated fatty acids chains and a decrease in C20 fatty acids in cancer cells compared with mammary epithelial cells was observed. At present, information about PL changes in cancer progression is lacking. Therefore, these data will be useful as a starting point to define possible PLs with prospective as biomarkers and disclose metabolic pathways with potential for therapy.
Alterations of phospholipid (PL) profiles have been associated to disease and specific lipids may be involved in the onset and evolution of cancer; yet, analysis of PL profiles using mass spectrometry (MS) in breast cancer cells is a novel approach. Previously, we reported a lipidomic analysis of PLs from mouse mammary epithelial and breast cancer cells using off-line thin layer chromatography (TLC)-MS, where several changes in PL profile were found to be associated with the degree of malignancy of cells. In the present study, lipidomic analysis has been extended to human mammary epithelial cells and breast cancer cell lines (MCF10A, T47-D, and MDA-MB-231), using TLC-MS, validated by hydrophilic interaction liquid chromatography-MS. Differences in phosphatidylethanolamine (PE) content relative to total amount of PLs was highest in non-malignant cells while phosphatidic acid was present with highest relative abundance in metastatic cells. In addition, the following differences in PL molecular species associated to cancer phenotype, metastatic potential, and cell morphology were found: higher levels of alkylacyl PCs and phosphatidylinositol (PI; 22:5/18:0) were detected in migratory cells, epithelial cells had less unsaturated fatty acyl chains and shorter aliphatic tails in PE and sphingomyelin classes, while PI (18:0/18:1) was lowest in non-malignant cells compared to cancer cells. To date, information about PL changes in cancer progression is scarce, therefore results presented in this work will be useful as a starting point to define possible PLs with prospective as biomarkers and disclose metabolic pathways with potential for cancer therapy.
Phosphatidylethanolamine glycation occurs in diabetic patients and was found to be related with oxidative stress and with diabetic complications. Glycated phosphatidylethanolamines seem to increase oxidation of other molecules; however, the reason why is not understood. In this work, we have studied the oxidation of glycated phosphatidylethanolamines (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylethanolamine (PLPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (dPPE)) using a Fenton system. Liquid chromatography-electrospray ionization (ESI)-mass spectrometry and ESI-tandem mass spectrometry in both positive and negative modes were used for detecting and identifying the oxidation products. We were able to identify several oxidation products with oxidation in unsaturated sn-2 acyl chain of PLPE, as long- and short-chain products with main oxidation sites on C-7, C-8, C-9, and C-12 carbons. Other products were identified in both glycated PLPE and glycated dPPE, revealing that oxidation also occurs in the glycated polar head. This fact has not been reported before. These products may be generated from oxidation of glycated phosphatidylethanolamines (PE) as Schiff base, leading to short-chain product without the amine moiety, due to cleavage of glycated polar head and long-chain product with two keto groups linked to the glycated polar head or from glycated PE as Amadori product, short-chain products with -NHCHO and -NHCHOHCHO terminal in polar head. Oxidation of glycated phosphatidylethanolamines occurred more quickly than the oxidation of non-glycated phosphatidylethanolamines probably because of the existence of more oxidation sites derived from glycation of polar head group. Monitoring glycated polar head oxidation could be important to evaluate oxidative stress modifications that occur in diabetic patients.
Phosphatidylethanolamines are a major class of phospholipids found in cellular membranes. Identification of the alterations in these phospholipids, induced by free radicals, could provide new tools for in vivo diagnosis of oxidative stress. In this study, 1-palmitoyl-2-linoleoyl-phosphatidylethanolamine oxidation products, induced by the hydroxyl radical, were studied using LC-MS and LC-MS/MS. Data obtained allowed the identification and separation of isomeric oxidative products with modifications in the sn-2 acyl chain, attributed to long- and short-chain products. Among long-chain products keto, keto-hydroxy, hydroxy, poly-hydroxy, peroxy and hydroxy-peroxy derivatives were identified. Product ions formed by loss of two H2O molecules vs loss of HOOH, allowed the identification of, respectively, di- (or poli-) hydroxy vs peroxy derivatives. Location of functional groups was determined by the product ions formed by cleavage of C-C bonds, in the vicinity of the oxidation positions, allowing the identification of C9, C12 and C13 as the predominant substituted positions. Short-chain products identified comprised aldehydes, hydroxy-aldehydes and carboxylic derivatives, with modified sn-2 acyl lengths of C7-C9 and C11, C12. Among the short-chain products identified, C9 products showed higher relative abundance.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractDepression is a worldwide disability disease associated with high morbidity and has increased dramatically in the last few years. The differential diagnosis and the definition of an individualized therapy for Depression is hampered by the absence of specific biomarkers. The aim of this study was to evaluate the phospholipdiomic profile of brain and myocardium in a mouse model of depression induced by chronic unpredictable stress. The lipidomic profile was evaluated by thin layer and liquid chromatography and mass spectrometry and lipid oxidation was estimated by FOX II assay. Antioxidant enzymes activity and the GSH/GSSG ratio were also evaluated.Results showed that chronic stress affect primarily the lipid profile of the brain, inducing an increased in lipid hydroperoxides, which was not detected in the myocardium. A significant decrease in phospahtidylinositol (PI) and in cardiolipin (CL) relative contents and also oxidation of cardiolipin and significant increase of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were observed in in brain from mice after unpredictable chronic stress conditions. In myocardium only an increase in PC content was observed. Nevertheless, both organs present a decreased GSH/GSSG ratio when compared to control groups, corroborating the occurrence of oxidative stress. The enzyme activities CAT and SOD were found to be decreased in the myocardium and increased in the brain, while glutathione reductase (GR) was decreased in brain. Our results indicate that in a mouse model for studying depression induced by chronic unpredictable stress, the modification of the expression of oxidative stress related enzymes did not prevent lipid oxidation in organs, particularly in the brain. These observations suggest that depression has an impact in the brain lipidome and that further studies are needed to better understand lipid role in depression and to evaluate their potential as future biomarkers.3
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